The overall goal of this proposal is to characterize a novel regulatory function of glycine N- methyltransferase (GNMT), an abundant metabolic enzyme. GNMT exists as a tetramer of four identical subunits and catalyses the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine producing sarcosine (N-methylglycine) and S-adenosylhomocysteine (SAH). It is believed that this reaction regulates the SAM/SAH ratio, which defines the methylation potential of the cell. GNMT is inhibited by 5-methyltetrahydrofolate, which provides a regulatory mechanism for the flow of methyl groups between the one-carbon folate pool and the methylation cycle. As such, enzyme catalysis, and an associated cellular role, is controlled to a significant extent by the availability of folate and methionine, which in turn depends on a dietary supply. Importantly, while GNMT is abundant in several human tissues, its expression is lost in tumors. We have also shown that the enzyme is not detectable in eight cancer cell lines tested so far. A possible explanation of this phenomenon came from our recent experiments, which demonstrated a function of the enzyme as a negative regulator of cellular proliferation. Overall, the following preliminary data, together with our published studies, laid the ground for the present application: (i) transient expression of GNMT suppresses proliferation of cancer cells;(ii) this effect cannot be reversed by high folate supplementation;(iii) PC3 cells activate the ERK pathway as a pro-survival mechanism in response to GNMT expression (but it is not sufficient to rescue cells);(iv) GNMT-sensitive cells revealed the presence of the protein in nuclei;(v) GNMT-resistant HEK293 cells did not accumulate the protein in nuclei;(v) in vitro chemical modification of lysine residues of GNMT causes dissociation of tetramer to monomers, followed by translocation of monomers into the nuclei. Accordingly, we hypothesize that GNMT controls cellular proliferation through regulation of gene expression. Specifically, we hypothesize that this role is not associated with the metabolic function of conversion of SAM to SAH but is exerted upon translocation into nuclei. We further propose that nuclear GNMT affects transcription through interaction with nuclear proteins/chromatin. We also propose that modifications of lysine residues on the interface of GNMT subunits within the enzyme tetramer results in dissociation to monomers and enables the nuclear entry. The specific aims designed to test these hypotheses are: (1) Characterize nuclear localization of GNMT. (2) Elucidate mechanisms promoting translocation of GNMT to the nucleus. (3) Differentiate the cytoplasm-related metabolic and nucleus-associated effects of GNMT in induction of cytotoxicity. Accomplishment of this project will establish a mechanism for GNMT nuclear regulation and define cellular processes controlled by the enzyme. PUBLIC HEALTH RELEVANCE: Folate, an important and essential part of the human diet, regulates many cellular processes including methylation, while folate deficiency promotes many diseases. This application is focused on a novel nucleus-associated function of GNMT, an abundant human enzyme regulated by folate, which lies at an intersection point between folate metabolism and methylation. Since GNMT can function as a restrictor of excessive proliferation in liver and other tissues, understanding its role in cellular metabolism will help to better manage liver related diseases.